VSMCs migration is essential in multiple disease processes, namely atherosclerosis, in-stent restenosis and transplant vasculopathy. Migration is a finely orchestrated process that involves the interaction of the extracellular matrix (ECM), integrins, cytoskeletal proteins and NADPH oxidases (Noxes). The first step in migration involves the cell sensing a gradient and establishing polarity;this is followed by lamellipodia extension. The VSMC then engages the ECM via integrin attachment and activation. This results in actin polymerization and formation of focal adhesions (FA). The cell uses the FA as a strut against which it contracts and moves. NADPH oxidases, by producing reactive oxygen species (O2-. and H2O2), regulate the process of migration. Different Noxes are present in different areas of the vasculature. In the large arteries, VSMCs contain Nox4 and Nox1. While Nox1 is located mainly in lamellipodia and caveolae, Nox4 is located in FA and in the nucleus. Knockdown of either Nox1 or Nox4 impairs VSMC migration, and knockdown of Nox4 impairs cell attachment and stress fiber formation, both processes in which integrins are critically important. This suggests that integrins may regulate migration and cytoskeletal integrity via Nox4 activation. Our objective is to elucidate the mechanism of activation of Nox4 by integrins after VSMC engagement of the ECM, and to define their roles in actin polymerization and migration. We will initially focus on integrin- activated, protein kinase C-mediated regulation of the Nox4 binding protein Poldip2. Experimental techniques will include genetic manipulation of Nox4 levels, measurement of reactive oxygen species, assessment of actin polymerization and binding to cortactin, an actin binding protein that regulates polymerization, visualization and quantification of focal adhesions and measurement of cell migration. We believe that elucidating the mechanism of activation of Nox4 after the integrins attach to the ECM will provide new targets of therapy for reducing or abolishing VSMCs migration. This would have tremendous impact on incidence of heart disease and in-stent restenosis after Percutaneous Coronary Intervention. The public health implications of new therapies aimed at reducing coronary artery disease incidence and prevalence are enormous. PUBLIC HEALTH RELEVANCE: Project Narrative Vascular smooth muscle cell (VSMC) migration plays a central role in atherosclerosis leading to progression to coronary heart disease. NADPH oxidases such as Nox4 are enzymes that fine tune the process of migration. Unraveling the proteins involved with Nox4 in orchestrating the process of migration will lead to better understanding of atherosclerosis and heart disease and help reduce the burden of this ever growing epidemic.